New biochemical methods for detection of misincorporation during DNA synthesis on natural templates in vitro will be developed. In one approach, attempts will be made to purify an endonuclease or N-glycosylase that specifically acts on DNA containing mismatched bases. If it can be isolated, this enzyme will be used to probe for misincorporation during DNA synthesis on a circular strand bearing a short primer. (If misincorporation occurred, the closed circular duplex product would contain mismatches, and thus be susceptible to cleavage by the enzyme). In a second approach, a short restriction fragment is used to prime DNA synthesis on a circular strand in the presence of only three of the dNTP's. Misincorporation allows extension of the primer past the point at which the missing nucleotide is normally required. Both these approaches will be used to screen for error-prone DNA polymerases, and to search for inducible activities that decrease the fidelity of DNA synthesis.